metallothionein and 3-nitrotyrosine

Oxidative stress mediated by hepatic CYP2E1 during isoniazid (INH) metabolism is considered responsible for INH hepatotoxicity. This study attempts to determine whether metallothionein (MT), a cysteine-rich antioxidant can protect against INH-induced liver injury by using a MT-I/II deficient mouse model (MT-/- mice). MT-/- mice and the corresponding wild-type mice received intragastric administrations of 0, 75, 150 and 300 mg/kg of INH for 15 days. The results showed that 150 and 300 mg/kg of INH induced liver injury in both types of mice, as evidenced by increased liver index and histopathological change of liver vacuolar degeneration. Increased hepatic MDA level and 3-NT expression, and decreased GSH content and SOD activity were also observed in both types of mice, indicating the involvement of oxidative and nitrosative stress. INH treatment upregulated hepatic CYP2E1 expression in both types of mice, and the severity of liver injury was in concert with the elevation of CYP2E1 expression. Comparative analyses revealed liver vacuolar degeneration and oxidative and nitrosative stress were more severe in MT-/- mice than wild-type mice, suggesting the hepatoprotection of MT against INH hepatotoxicity. Taken together, these findings clearly demonstrate that MT protects against INH-induced liver toxicity by ameliorating CYP2E1-dependent oxidative and nitrosative impairment.

Topics: Animals; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP2E1; Isoniazid; Lipid Peroxidation; Liver; Male; Metallothionein; Mice, Inbred C57BL; Mice, Mutant Strains; Oxidative Stress; Tyrosine

Epilepsy is a neurological disorder that has been associated with oxidative stress therefore epilepsy models have been develop such as kainic acid and pentylenetetrazol are usually used to understanding of the molecular mechanisms of this disease. We examined the metallothionein expression in rat brains of treated with kainic acid and pentylenetetrazol. Increase in metallothionein and nitrotirosyne immunoreactivity of both seizures epilepsy models was observed. Moreover, we show a significant increase on levels of MT expression. These results suggest that the increase of metallothionein expression is related with kainic acid and pentylenetetrazol treatments as response to damage mediated by oxidative stress.

Topics: Animals; Brain; Disease Models, Animal; Epilepsy; Kainic Acid; Male; Metallothionein; Oxidative Stress; Pentylenetetrazole; Rats; Rats, Wistar; Tyrosine

The present study was to explore the effect of metallothionein (MT) on intermittent hypoxia (IH) induced aortic pathogenic changes. Markers of oxidative damages, inflammation, and vascular remodeling were observed by immunohistochemical staining after 3 days and 1, 3, and 8 weeks after IH exposures. Endogenous MT was induced after 3 days of IH but was significantly decreased after 8 weeks of IH. Compared with the wild-type mice, MT knock-out mice exhibited earlier and more severe pathogenic changes of oxidative damages, inflammatory responses, and cellular apoptosis, as indicated by the significant accumulation of collagen, increased levels of connective tissue growth factor, transforming growth factor β1, tumor necrosis factor-alpha, vascular cell adhesion molecule 1,3-nitrotyrosine, and 4-hydroxy-2-nonenal in the aorta. These findings suggested that chronic IH may lead to aortic damages characterized by oxidative stress and inflammation, and MT may play a pivotal role in the above pathogenesis process.

Topics: Aldehydes; Animals; Aorta; Apoptosis; Connective Tissue Growth Factor; Hypoxia; Metallothionein; Mice; Mice, Knockout; NADPH Oxidases; Nitric Oxide Synthase Type III; Oxidative Stress; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine

We previously demonstrated that metallothionein (MT)-mediated protection from diabetes-induced pathological changes in cardiac tissues is related to suppression of superoxide generation and protein nitration. The present study investigated which diabetes-nitrated protein(s) mediate the development of these pathological changes by identifying the panel of nitrated proteins present in diabetic hearts of wild-type (WT) mice and not in those of cardiac-specific MT-overexpressing transgenic (MT-TG) mice. At 2, 4, 8, and 16 wk after streptozotocin induction of diabetes, histopathological examination of the WT and MT-TG diabetic hearts revealed cardiac structure derangement and remodeling, significantly increased superoxide generation, and 3-nitrotyrosine accumulation. A nitrated protein of 58 kDa, succinyl-CoA:3-ketoacid CoA transferase-1 (SCOT), was identified by mass spectrometry. Although total SCOT expression was not significantly different between the two types of mice, the diabetic WT hearts showed significantly increased nitration content and dramatically decreased catalyzing activity of SCOT. Although SCOT nitration sites were identified at Tyr(76), Tyr(117), Tyr(135), Tyr(226), Tyr(368), and Trp(374), only Tyr(76) and Trp(374) were found to be located in the active site by three-dimensional structure modeling. However, only Trp(374) showed a significantly different nitration level between the WT and MT-TG diabetic hearts. These results suggest that MT prevention of diabetes-induced pathological changes in cardiac tissues is most likely mediated by suppression of SCOT nitration at Trp(374).

Topics: Animals; Coenzyme A-Transferases; Diabetes Mellitus, Experimental; Disease Models, Animal; Energy Metabolism; Heart Diseases; Male; Metallothionein; Mice; Mice, Inbred Strains; Mice, Transgenic; Myocardium; Nitrogen; Protein Structure, Tertiary; Superoxides; Tryptophan; Tyrosine

Primary central nervous system lymphoma (PCNSL) in immunocompetent patients is highly malignant and has a poor prognosis. The PCNSL molecular features are reminiscent to some degree of diffuse large B-cell lymphoma (DLBCL), yet PCNSL shows unique molecular profiles and a distinct clinical behavior. This article characterizes the histopathology and expression profiles of metallothionein-I + II (MT-I + II) and their receptor megalin along with proliferation, oxidative stress, and apoptosis in PCNSL and in central nervous system (CNS) lymphomas due to relapse from DLBCL (collectively referred to as CNS lymphoma). We show for the first time that MT-I + II and megalin are significantly altered in CNS lymphoma relative to controls (reactive lymph nodes and non-lymphoma brain tissue with neuropathology). MT-I + II are secreted in the CNS and are found mainly in the lymphomatous cells, while their receptor megalin is increased in cerebral cells. This morphology likely reflects the CNS lymphoma microenvironment and molecular interactions between lymphomatous and neuronal cells.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Brain; Cell Cycle Proteins; Cell Proliferation; Central Nervous System Neoplasms; DNA-Binding Proteins; Guanine; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Low Density Lipoprotein Receptor-Related Protein-2; Lymph Nodes; Lymphoma, Large B-Cell, Diffuse; Malondialdehyde; Metallothionein; Middle Aged; Minichromosome Maintenance Complex Component 7; Nuclear Proteins; Oxidative Stress; Tyrosine

A dyshomeostasis of extra- and intracellular Ca(2+) and Zn(2+) occurs in rats receiving chronic aldosterone/salt treatment (ALDOST). Herein, we hypothesized that the dyshomeostasis of intracellular Ca(2+) and Zn(2+) is intrinsically coupled that alters the redox state of cardiac myocytes and mitochondria, with Ca(2+) serving as a pro-oxidant and Zn(2+) as an antioxidant. Toward this end, we harvested hearts from rats receiving 4 weeks of ALDOST alone or cotreatment with either spironolactone (Spiro), an aldosterone receptor antagonist, or amlodipine (Amlod), an L-type Ca(2+) channel blocker, and from age/sex-matched untreated controls. In each group, we monitored cardiomyocyte [Ca(2+)]i and [Zn(2+)]i and mitochondrial [Ca(2+)]m and [Zn(2+)]m; biomarkers of oxidative stress and antioxidant defenses; expression of Zn transporters, Zip1 and ZnT-1; metallothionein-1, a Zn(2+)-binding protein; and metal response element transcription factor-1, a [Zn(2+)]i sensor and regulator of antioxidant defenses. Compared with controls, at 4-week ALDOST, we found the following: (a) increased [Ca(2+)]i and [Zn(2+)]i, together with increased [Ca(2+)]m and [Zn(2+)]m, each of which could be prevented by Spiro and attenuated with Amlod; (b) increased levels of 3-nitrotyrosine and 4-hydroxy-2-nonenal in cardiomyocytes, together with increased H(2)O(2) production, malondialdehyde, and oxidized glutathione in mitochondria that were coincident with increased activities of Cu/Zn superoxide dismutase and glutathione peroxidase; and (c) increased expression of metallothionein-1, Zip1 and ZnT-1, and metal response element transcription factor-1, attenuated by Spiro. Thus, an intrinsically coupled dyshomeostasis of intracellular Ca(2+) and Zn(2+) occurs in cardiac myocytes and mitochondria in rats receiving ALDOST, where it serves to alter their redox state through a respective induction of oxidative stress and generation of antioxidant defenses. The importance of therapeutic strategies that can uncouple these two divalent cations and modulate their ratio in favor of sustained antioxidant defenses is therefore suggested.

Topics: Aldehydes; Aldosterone; Amlodipine; Animals; Calcium; Calcium Channel Blockers; Chronic Disease; Disease Models, Animal; Glutathione Peroxidase; Homeostasis; Hydrogen Peroxide; Hyperaldosteronism; Male; Metallothionein; Mineralocorticoid Receptor Antagonists; Mitochondria, Heart; Myocytes, Cardiac; Oxidative Stress; Rats; Rats, Sprague-Dawley; Spironolactone; Superoxide Dismutase; Tyrosine; Zinc

Metallothionein (MT) has been shown to be an effective protector against DOX-induced cardiomyopathy, however the involved precise mechanisms are still unknown. The present study was undertaken to clarify whether the inhibition of superoxide generation and related nitrosative damage were involved in the metallothionein attenuation of DOX-induced cardiac injury. MT-I/II null (MT-/-) mice and corresponding wild-type mice (MT+/+) were pretreated with either saline or zinc (300 micromol/kg, s.c., once a day for 2 days) prior to a single dose of DOX (15 mg/kg, i.p.) or equal volume of saline. Animals were sacrificed on the 4th day after DOX administration and samples were collected for further analyses. DOX caused remarkable cardiac damage in both MT+/+ and MT-/- mice as demonstrated by biochemical and histopathological alterations. Zinc pretreatment significantly increased the cardiac MT levels and therefore inhibited the cardiac toxic effects of DOX only in MT+/+ mice, but not in MT-/- mice. Furthermore, elevated formation of superoxide and peroxynitrite were obviously observed after DOX treatment, while these elevation were prevented by MT induction by zinc in MT+/+ mice, but not in MT-/- mice. These findings suggest that metallothionein induction by zinc exhibits protective effects on the cardiac toxicology of DOX, which might be mediated through the prevention of superoxide generation and related nitrosative impairment.

Topics: Animals; Antibiotics, Antineoplastic; Doxorubicin; Heart; Immunohistochemistry; Male; Metallothionein; Mice; Mice, Knockout; Myocardium; Peroxynitrous Acid; Superoxides; Troponin T; Tyrosine; Zinc

Senescence is accompanied by oxidative stress and cardiac dysfunction, although the link between the two remains unclear. This study examined the role of antioxidant metallothionein on cardiomyocyte function, superoxide generation, the oxidative stress biomarker aconitase activity, cytochrome c release, and expression of oxidative stress-related proteins, such as the GTPase RhoA and NADPH oxidase protein p47phox in young (5-6 mo) and aged (26-28 mo) FVB wild-type (WT) and cardiac-specific metallothionein transgenic mice. Metallothionein mice showed a longer life span (by approximately 4 mo) than FVB mice evaluated by the Kaplan-Meier survival curve. Compared with young cardiomyocytes, aged myocytes displayed prolonged TR(90), reduced tolerance to high stimulus frequency, and slowed intracellular Ca2+ decay, all of which were nullified by metallothionein. Aging increased superoxide generation, active RhoA abundance, cytochrome c release, and p47phox expression and suppressed aconitase activity without affecting protein nitrotyrosine formation in the hearts. These aging-induced changes in oxidative stress and related protein biomarkers were attenuated by metallothionein. Aged metallothionein mouse myocytes were more resistant to the superoxide donor pyrogallol-induced superoxide generation and apoptosis. In addition, aging-associated prolongation in TR90 was blunted by the Rho kinase inhibitor Y-27632. Collectively, our data demonstrated that metallothionein may alleviate aging-induced cardiac contractile defects and oxidative stress, which may contribute to prolonged life span in metallothionein transgenic mice.

Topics: Aconitate Hydratase; Aging; Animals; Apoptosis; Calcium; Cytochromes c; Diastole; Gene Expression Regulation; Heart Ventricles; Metallothionein; Mice; Mice, Transgenic; Myocardial Contraction; Myocytes, Cardiac; Oxidative Stress; Pyrogallol; rhoA GTP-Binding Protein; Superoxides; Tyrosine

We aimed to test whether attenuation of early-phase cardiac cell death can prevent diabetic cardiomyopathy.. Our previous study showed that cardiac apoptosis as a major early cellular response to diabetes is induced by hyperglycemia-derived oxidative stress that activates a mitochondrial cytochrome c-mediated caspase-3 activation pathway. Metallothionein (MT) as a potent antioxidant prevents the development of diabetic cardiomyopathy.. Diabetes was induced by a single dose of streptozotocin (STZ) (150 mg/kg) in cardiac-specific, metallothionein-overexpressing transgenic (MT-TG) mice and wild-type (WT) controls. On days 7, 14, and 21 after STZ treatment, cardiac apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activation. Cardiomyopathy was evaluated by cardiac ultrastructure and fibrosis in the diabetic mice 6 months after STZ treatment.. A significant reduction in diabetes-induced increases in TUNEL-positive cells, caspase-3 activation, and cytochrome c release from mitochondria was observed in the MT-TG mice as compared to WT mice. Cardiac protein nitration (3-nitrotyrosine [3-NT]) and lipid peroxidation were significantly increased, and there was an increase in mitochondrial oxidized glutathione and a decrease in mitochondrial reduced glutathione in the WT, but not in the MT-TG, diabetic mice. Double staining for cardiomyocytes with alpha sarcomeric actin and caspase-3 or 3-NT confirmed the cardiomyocyte-specific effects. A significant prevention of diabetic cardiomyopathy and enhanced animal survival were observed in the MT-TG diabetic mice as compared to WT diabetic mice.. These results suggest that attenuation of early-phase cardiac cell death by MT results in a significant prevention of the development of diabetic cardiomyopathy. This process is mediated by MT suppression of mitochondrial oxidative stress.

Topics: Actins; Animals; Antioxidants; Apoptosis; Cardiomyopathies; Caspase 3; Cell Death; Cytochromes c; Diabetes Mellitus, Experimental; Enzyme Activation; Glutathione; Glutathione Disulfide; Lipid Peroxidation; Metallothionein; Mice; Mice, Transgenic; Mitochondria, Heart; Myocardium; Myocytes, Cardiac; Oxidative Stress; Sarcomeres; Survival Analysis; Tyrosine

We examined metallothionein (MT)-induced neuroprotection during kainic acid (KA)-induced excitotoxicity by studying transgenic mice with MT-I overexpression (TgMT mice). KA induces epileptic seizures and hippocampal excitotoxicity, followed by inflammation and delayed brain damage. We show for the first time that even though TgMT mice were more susceptible to KA, the cerebral MT-I overexpression decreases the hippocampal inflammation and delayed neuronal degeneration and cell death as measured 3 days after KA administration. Hence, the proinflammatory responses of microglia/macrophages and lymphocytes and their expression of interleukin (IL)-1, IL-6, IL-12, tumor necrosis factor-alpha and matrix metalloproteinases (MMP-3, MMP-9) were significantly reduced in hippocampi of TgMT mice relative to wild-type mice. Also by 3 days after KA, the TgMT mice showed significantly less delayed damage, such as oxidative stress (formation of nitrotyrosine, malondialdehyde, and 8-oxoguanine), neurodegeneration (neuronal accumulation of abnormal proteins), and apoptotic cell death (judged by TUNEL and activated caspase-3). This reduced bystander damage in TgMT mice could be due to antiinflammatory and antioxidant actions of MT-I but also to direct MT-I effects on the neurons, in that significant extracellular MT presence was detected. Furthermore, MT-I overexpression stimulated astroglia and increased immunostaining of antiinflammatory IL-10, growth factors, and neurotrophins (basic fibroblastic growth factor, transforming growth factor-beta, nerve growth factor, brain-derived neurotrophic factor, glial-derived neurotrophic factor) in hippocampus. Accordingly, MT-I has different functions that likely contribute to the increased neuron survival and improved CNS condition of TgMT mice. The data presented here add new insight into MT-induced neuroprotection and indicate that MT-I therapy could be used against neurological disorders.

Topics: Amyloid beta-Peptides; Analysis of Variance; Animals; Astrocytes; Cell Count; Cell Death; Central Nervous System Diseases; Epilepsy; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Growth Substances; Guanine; Hippocampus; Immunohistochemistry; In Situ Nick-End Labeling; Interleukins; Kainic Acid; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurodegenerative Diseases; Neurofibrillary Tangles; Staining and Labeling; Tyrosine

Metallothionein (MT) is a protein that can be induced by inflammatory mediators and participates in cytoprotection. However, its role in antigen-related inflammation remains to be established. We determined whether intrinsic MT protects against antigen-related airway inflammation induced by ovalbumin (OVA) in MT-I/II null (MT [-/-]) mice and in corresponding wild-type (WT) mice. MT (-/-) mice and WT mice were intratracheally challenged with OVA (1 mug per body) biweekly four times. Twenty-four hours after the last OVA challenge, significant increases were shown in the numbers of total cells, eosinophils, and neutrophils in bronchoalveolar lavage fluid from MT (-/-) mice than in those from WT mice. The protein level of interleukin-1beta (IL-1beta) was significantly greater in MT (-/-) mice than in WT mice after OVA challenge. Immunohistochemical analysis showed that the formations of 8-oxy-deoxyguanosine and nitrotyrosine in the lung were more intense in MT (-/-) mice than in WT mice after OVA challenge. These results indicate that endogenous MT is a protective molecule against antigen-related airway inflammation induced by OVA, at least partly, via the suppression of enhanced lung expression of IL-1beta and via the antioxidative properties. Our findings suggest that MT may be a therapeutic target for the treatment of antigen-related airway inflammatory diseases such as bronchial asthma.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antigens; Bronchitis; Bronchoalveolar Lavage Fluid; Chemokines; Cytokines; Deoxyguanosine; Immunohistochemistry; Metallothionein; Mice; Trachea; Tyrosine

Oxygen free radicals and nitric oxide (NO) participate in the pathogenesis of acute central nervous system (CNS) injury by forming peroxynitrite, which promotes oxidative damage and tyrosine nitration. Neuronal nitration is associated with cell death, but little is known of the characteristics and cell fate of nitrated astrocytes. In this study, we have used a postnatal excitotoxic lesion model (intracortical NMDA injection) and our aims were (i) to evaluate the temporal and spatial pattern of astroglial nitration in correlation with the neuropathological process and the sources of NO; and (ii) to establish, if any, the correlation among astrocyte nitration and other events such as expression of cytoskeletal proteins, antioxidant enzymes, and cell death markers to cope with nitration and/or undergo cell death. Our results show that after postnatal excitotoxic damage two distinct waves of nitration were observed in relation to astrocytes. At 24 h post-lesion, early-nitrated astrocytes were found within the neurodegenerating area, coinciding with the time of maximal cell death. These early-nitrated astrocytes are highly ramified protoplasmic cells, showing diffuse glial fibrillary acidic protein (GFAP) content and expressing inducible NOS. At later time-points, when astrogliosis is morphologically evident, nitrated hypertrophied reactive astrocytes are observed in the penumbra and the neurodegenerated area, displaying increased expression of GFAP and vimentin cytoskeletal proteins and of metallothionein I-II and Cu/Zn superoxide dismutase antioxidant proteins. Moreover, despite revealing activated caspase-3, they do not show TUNEL labeling. In summary, we show that nitrated astrocytes in vivo constitute a subpopulation of highly reactive astrocytes which display high resistance towards oxidative stress induced cell death.

Topics: Animals; Animals, Newborn; Astrocytes; Brain Injuries; Cytoskeletal Proteins; Disease Models, Animal; Female; Male; Metallothionein; Nerve Tissue Proteins; Neurotoxins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Rats; Rats, Long-Evans; Superoxide Dismutase; Tyrosine

The mechanisms of metallothionein prevention of diabetic cardiomyopathy are largely unknown. The present study was performed to test whether inhibition of nitrosative damage is involved in metallothionein prevention of diabetic cardiomyopathy. Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice and wild-type littermate controls were treated with streptozotocin (STZ) by a single intraperitoneal injection, and both developed diabetes. However, the development of diabetic cardiomyopathy, revealed by histopathological and ultrastructural examination, serum creatine phosphokinase, and cardiac hemodynamic analysis, was significantly observed only in the wild-type, but not in MT-TG, diabetic mice 2 weeks and 6 months after STZ treatment. Formations of superoxide and 3-nitrotyrosine (3-NT), a marker for peroxynitrite-induced protein damage, were detected only in the heart of wild-type diabetic mice. Furthermore, primary cultures of cardiomyocytes from wild-type and MT-TG mice were exposed to lipopolysaccharide/tumor necrosis factor-alpha for generating intracellular peroxynitrite. Increases in 3-NT formation and cytotoxicity were observed in wild-type, but not in MT-TG, cardiomyocytes. Either urate, a peroxynitrite-specific scavenger, or Mn(111) tetrakis 1-methyl 4-pyridyl porphyrin pentachloride (MnTMPyP), a superoxide dismutase mimic, significantly inhibited the formation of 3-NT along with a significant prevention of cytotoxicity. These results thus suggest that metallothionein prevention of diabetic cardiomyopathy is mediated, at least in part, by suppression of superoxide generation and associated nitrosative damage.

Topics: Animals; Cardiomyopathies; Diabetes Complications; Gene Expression; Heart Diseases; Male; Metallothionein; Mice; Mice, Transgenic; Nitrosation; Superoxides; Tyrosine

Oxidative stress is involved in the pathogenesis of diabetes and its cardiovascular complications. Metallothionein (MT), a stress-response protein, is significantly increased in the liver and kidney of diabetic animals. We examined whether diabetes also induces cardiac MT synthesis through oxidative damage and whether MT overexpression protects the heart from injury. Diabetes was induced in mice by single injection of streptozotocin (STZ), and cardiac MT mRNA and protein levels were measured 2 weeks and 2 months after STZ treatment. Diabetes significantly increased cardiac MT synthesis 2 weeks and 2 months after STZ treatment, with no change in cardiac metals including zinc, copper, and iron. Serum and cardiac vasopeptide endothelin and inflammatory cytokine tumor necrosis factor-alpha were also significantly increased in diabetic hearts, as were the ratio of oxidized to reduced glutathione and the immunohistochemical staining of 3-nitrotyrosine and 4-hydroxynonenal. To explore the biological importance of increased MT synthesis in the heart, MT-overexpressing transgenic mice were treated with STZ and then examined 2 months later. A loss of inotropic reserve, uncovered during beta-adrenergic stimulation, and the presence of cardiac fibrosis, shown by increased Sirius red staining of collagen, were evident in the wild-type diabetic mice but not in the MT-overexpressing transgenic diabetic mice. These results suggest that diabetes-induced cardiac MT expression likely associates with systemic increases in endothelin-1 and tumor necrosis factor-alpha and the resulting cardiac oxidative stress. Overexpressing cardiac MT significantly protects the heart from diabetes-induced injury.

Topics: Aldehydes; Animals; Blotting, Northern; Blotting, Western; Cardiovascular Diseases; Copper; Diabetes Mellitus, Experimental; Endothelin-1; Glutathione; Immunohistochemistry; Interleukin-6; Iron; Metallothionein; Mice; Mice, Transgenic; Myocardium; Oxidative Stress; RNA, Messenger; Tumor Necrosis Factor-alpha; Tyrosine; Zinc

6-aminonicotinamide (6-AN) is a niacin antagonist, which leads to degeneration of gray matter astrocytes mainly in the brainstem. We have examined the role of interleukin-6 (IL-6) in this degenerative process by using transgenic mice with astrocyte-targeted IL-6 expression (GFAP-IL6 mice). This study demonstrates that transgenic IL-6 expression significantly increases the 6-AN-induced inflammatory response of reactive astrocytes, microglia/macrophages, and lymphocytes in the brainstem. Also, IL-6 induced significant increases in proinflammatory cytokines IL-1, IL-12, and tumor necrosis factor-alpha as well as growth factors basic fibroblast growth factor (bFGF), transforming growth factor-beta, neurotrophin-3, angiopoietin, vascular endothelial growth factor, and the receptor for bFGF. In accordance, angiogenesis was increased in GFAP-IL6 mice relative to controls after 6-AN. Moreover, oxidative stress and apoptotic cell death were significantly reduced by transgenic IL-6 expression. IL-6 is also a major inducer in the CNS of metallothionein I and II (MT-I+II), which were significantly increased in the GFAP-IL6 mice. MT-I+II are antioxidants and neuroregenerative factors in the CNS, so increased MT-I+II levels in GFAP-IL6 mice could contribute to the reduction of oxidative stress and cell death in these mice.

Topics: 6-Aminonicotinamide; Angiogenesis Inducing Agents; Animals; Apoptosis; Astrocytes; Brain Stem; Cell Count; Central Nervous System; Cytokines; Disease Models, Animal; Gene Targeting; Glial Fibrillary Acidic Protein; Growth Substances; Immunohistochemistry; In Situ Nick-End Labeling; Interleukin-6; Lymphocytes; Macrophages; Malondialdehyde; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Nerve Degeneration; Oxidative Stress; Staining and Labeling; Stem Cells; Teratogens; Tyrosine

Metallothionein-I+II (MT-I+II) are antioxidant, neuroprotective proteins, and in this report we have examined their roles during experimental autoimmune encephalomyelitis (EAE) by comparing MT-I+II-knock-out (MTKO) and wild-type mice. We herewith show that EAE susceptibility is higher in MTKO mice relatively to wild-type mice, and that the inflammatory responses elicited by EAE in the central nervous system (CNS) are significantly altered by MT-I+II deficiency. Thus, during EAE the MTKO mice showed increased macrophage and T-lymphocytes infiltration in the CNS, while their reactive astrogliosis was significantly decreased. In addition, the expression of the proinflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha elicited by EAE was further increased in the MTKO mice, and oxidative stress and apoptosis were also significantly increased in MTKO mice compared to normal mice. The present results strongly suggest that MT-I+II are major factors involved in the inflammatory response of the CNS during EAE and that they play a neuroprotective role in this scenario.

Topics: Animals; Apoptosis; Encephalomyelitis, Autoimmune, Experimental; Gliosis; In Situ Nick-End Labeling; Interleukin-1; Interleukin-6; Macrophages; Malondialdehyde; Metallothionein; Mice; Mice, Inbred Strains; Mice, Knockout; Microglia; Nerve Degeneration; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; T-Lymphocytes; Tumor Necrosis Factor-alpha; Tyrosine

We examined the effects of interleukin-6 (IL-6) deficiency on brain inflammation and the accompanying bone marrow (BM) leukopoiesis and spleen immune reaction after systemic administration of a niacin antagonist, 6-aminonicotinamide (6-AN), which causes both astroglial degeneration/cell death in brain stem gray matter areas and BM toxicity. In both normal and genetically IL-6-deficient mice (IL-6 knockout (IL-6KO) mice), the extent of astroglial degeneration/cell death in the brain stem was similar as determined from disappearance of GFAP immunoreactivity. In 6-AN-injected normal mice reactive astrocytosis encircled gray matter areas containing astroglial degeneration/cell death, which were infiltrated by several macrophages and some T-lymphocytes. Reactive astrocytes and a few macrophages increased significantly the antioxidants metallothionein-I+II (MT-I+II) and moderately the MT-III isoform. In 6-AN-injected IL-6KO mice reactive astrocytosis and recruitment of macrophages and T-lymphocytes were clearly reduced, as were BM leukopoiesis and spleen immune reaction. Expression of MT-I+II was significantly reduced while MT-III was increased. Oxidative stress, as determined by measuring nitrated tyrosine and malondialdehyde, was increased by 6-AN to a greater extent in IL-6KO mice. The blood-brain barrier to albumin was only disrupted in 6-AN-injected normal mice, which likely is due to the substantial migration of blood-derived inflammatory cells into the CNS. The present results demonstrate that inflammation in CNS is clearly reduced during IL-6 deficiency and this effect is likely due to significant inhibition of BM leukopoiesis. We also show that IL-6 deficiency reduces the levels of neuroprotective antioxidants MT-I+II followed by an increased oxidative stress during CNS inflammation.

Topics: 6-Aminonicotinamide; Animals; Antigens, Differentiation; Astrocytes; Blood-Brain Barrier; Bone Marrow Cells; Brain Stem; Encephalitis; Glial Fibrillary Acidic Protein; Hematopoiesis; Immunohistochemistry; Interleukin-6; Macrophages; Malondialdehyde; Metallothionein; Metallothionein 3; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Nerve Tissue Proteins; Oxidative Stress; Protein Isoforms; Serum Albumin; T-Lymphocytes; Tyrosine